Molecular mechanism of peptide-specific pheromone signaling in Enterococcus faecalis: functions of pheromone receptor TraA and pheromone-binding protein TraC encoded by plasmid pPD1.
Conjugative transfer of the Enterococcus faecalis plasmid pPD1 is activated by cPD1, one of several peptide sex pheromones secreted by plasmid-free recipient cells, and is blocked by a donor-produced peptide inhibitor, iPD1. Using a tritiated pheromone, [3H]cPD1, we investigated how pPD1-harboring donor cells receive these peptide signals. Donor cells rapidly incorporated [3H]cPD1. The cell extract but not the membrane fraction of the donor strain exhibited significant [3H]cPD1-binding activity. On the basis of these data and those of tracer studies, it was demonstrated that cPD1 was internalized, where it bound to a high-molecular-weight compound. The cell extract of a strain carrying the traA-bearing multicopy plasmid (pDLHH21) also exhibited high [3H]cPD1-binding activity. A recombinant TraA exhibited a dissociation constant of 0.49 +/- 0.08 nM against [3H]cPD1. iPD1 competitively inhibited [3H]cPD1 binding to TraA, whereas pheromones and inhibitors relating to other plasmid systems did not. These results show that TraA is a specific intracellular receptor for cPD1 and that iPD1 acts as an antagonist for TraA. A strain carrying the traC-bearing multicopy plasmid (pDLES23) exhibited significant [3H]cPD1-binding activity. A strain carrying traC-disrupted pPD1 (pAM351CM) exhibited lower [3H] cPD1-binding activity as well as lower sensitivity to cPD1 than a wild-type donor strain. Some of the other pheromones and inhibitors inhibited [3H]cPD1 binding to the traC transformant like cPD1 and iPD1 did. These results show that TraC, as an extracellular less-specific pheromone-binding protein, supports donor cells to receive cPD1.
Nakayama
J
J
Department of Applied Biological Chemistry, Graduate School of Agriculture and Life Sciences, University of Tokyo, Japan. ajiro@hongo.ecc.u-tokyo.ac.jp
Takanami
Y
Y
Horii
T
T
Sakuda
S
S
Suzuki
A
A
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D001667:Binding, Competitive; D002457:Cell Extracts; D002462:Cell Membrane / Q000378:metabolism; D013293:Enterococcus faecalis / Q000235:genetics / Q000378:metabolism*; D036781:Fimbriae Proteins; D016368:Frameshift Mutation; D009842:Oligopeptides / Q000096:biosynthesis / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism*; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013104:Spheroplasts / Q000378:metabolism; D013997:Time Factors; D014316:Tritium
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002457:Cell Extracts; D009842:Oligopeptides; D010675:Pheromones; D011993:Recombinant Fusion Proteins; C043210:Streptococcus faecalis sex pheromone cPD1; C051668:sex pheromone inhibitor iPD1; C074790:traC protein, Plasmid F; D014316:Tritium; D036781:Fimbriae Proteins